ORGANIC LETTERS
Microwave-Accelerated SPOT-Synthesis on Cellulose Supports
2004 Vol. 6, No. 12 2019-2022
Matthew D. Bowman, Ryan C. Jeske, and Helen E. Blackwell* Department of Chemistry, UniVersity of WisconsinsMadison, 1101 UniVersity AVenue, Madison, Wisconsin 53706-1322
[email protected] Received April 14, 2004
ABSTRACT
We demonstrate that microwave irradiation can dramatically accelerate reaction rates for spatially addressable library synthesis on planar membrane supports. The development of a robust support/linker system, microwave-assisted synthesis of small molecule test libraries, and methods for solid-phase scale-up on cellulose are described.
Combinatorial chemistry has become an important tool in many areas of research, including chemical biology and drug discovery.1 As the demand for libraries of small organic molecules continues to grow, particularly due to the burgeoning fields of genomics and proteomics, the need to develop new synthesis platforms for the rapid generation and evaluation of “natural product-based” and “drug-like” compounds becomes more urgent.2 Parallel and split-pool synthesis on polymeric solid supports are currently the most common strategies for the generation of combinatorial libraries.3 While both of these approaches have enabled the synthesis of large libraries, performing reactions on conventional solid-phase resins can have significant drawbackss the hydrophobic beads used are often expensive, difficult to manipulate, fragile, and not amenable to many types of biological assays. Furthermore, solid-phase reaction rates are frequently 10- to 100-fold slower than their solution-phase
counterparts.4 This problem frequently outweighs the purification benefits permitted by solid supports and requires significant time investment in reaction optimization. The development of an inexpensive and robust library synthesis platform that combines the purification benefits of solidphase resins with enhanced reaction rates would impact significantly the role of combinatorial techniques in research today. Here, we demonstrate that the strategic combination of microwave-assisted organic reactions with SPOT-synthesis on planar supports presents a platform that can address many of these current limitations.5 SPOT-synthesis is a conceptually simple and flexible approach for parallel library synthesis on planar cellulose supports (Figure 1).6 This method involves spatially addressed, solid-phase synthesis on derivatized cellulose sheets (readily prepared from inexpensive filter paper) to generate arrays of unique molecules (1-10,000 SPOTs).7,8 In contrast
* To whom correspondence should be addressed. (1) (a) Ley, S. V.; Baxendale, I. R. Nat. ReV. Drug. DiscoV. 2002, 1, 573-586. (b) Lam, K. S.; Renil, M. Curr. Opin. Chem. Biol. 2002, 6, 353358. (c) Erhardt, P. W. Pure Appl. Chem. 2002, 74, 703-785. (2) (a) Schreiber, S. L. Science 2000, 287, 1964-1969. (c) Seneci, P.; Miertus, S. Mol. DiVers. 2000, 5, 75-89. (3) (a) Boyle, N. A.; Janda, K. D. Curr. Opin. Chem. Biol. 2002, 6, 339346. (b) Tan, D. S.; Burbaum, J. J. Curr. Opin. Drug Discuss. DeVel. 2000, 3, 439-453.
(4) Li, W. B.; Yan, B. J. Org. Chem. 1998, 63, 4092-4097. (5) For recent commentaries on microwave-assisted combinatorial chemistry, see: (a) Blackwell, H. E. Org. Biomol. Chem. 2003, 1, 1251-1255. (b) Kappe, C. O. Curr. Opin. Chem. Biol. 2002, 6, 314-320. (6) Frank, R. Tetrahedron 1992, 48, 9217-9232. (7) For a recent SPOT-synthesis review, see: Frank, R. J. Immunol. Methods 2002, 267, 13-26. (8) We and others use Whatman 1Chr chromatography paper for SPOTsynthesis (thickness ) 0.34 mm and cost ca. 0.5¢/cm2).
10.1021/ol049313f CCC: $27.50 Published on Web 05/18/2004
© 2004 American Chemical Society
We initiated our investigations through the development of a robust, linker-functionalized planar cellulose support (Scheme 1). Previous cellulose derivatization methods for
Scheme 1.
Support Modification and Linker Installation
Figure 1. Schematic of the SPOT-synthesis process.
to conventional polystyrene resins, the hydrophilic, mechanically robust membrane sheets are easy to manipulate during synthesis and washing steps. Furthermore, cellulose membranes are compatible with various on-support biological screening methods, including protein-binding assays, enzymelinked immunosorbent assays (ELISA), and agar overlay.7 Despite the advantages outlined above, the application of the SPOT-synthesis technique has been largely restricted to the construction of simple peptide libraries.7 Examples of nonpeptide libraries generated via SPOT-synthesis are scarce, and most have been based on simple acylation chemistry.9 More challenging chemistry has not been pursued, in part, because (1) SPOT-synthesis suffers from slow reaction rates similar to conventional solid-phase synthesis and (2) general, reproducible methods for heating spatially addressed reactions are not available. We hypothesized that microwave (MW) assistance could broaden the reaction scope of SPOTsynthesis and facilitate the generation of complex small molecule libraries. The use of MW irradiation as a nonconventional heating method for organic synthesis has increased over the past decade, primarily due to marked reductions in reaction times and enhancements in conversion and purity.10 To date, however, the generality and utility of MW-assisted reactions on planar polymeric supports have not been explored.5a,11 We chose to examine the scope and limitations of MWassisted SPOT-synthesis through the preparation of libraries of chalcone-derived molecules, as chalcone synthesis via Claisen-Schmidt condensation has been performed with success in solution under MW conditions previously.10,12 Chalcones can be derivatized easily to generate diverse second-generation libraries.13,14 Further, chalcones present a proven molecular scaffold for biological evaluation, as certain derivatives exhibit potent and specific activities, e.g., activation of Sirtuin proteins15 and broad anticancer activity.16 (9) For selected examples, see: (a) Scharn, D.; Germeroth, L.; SchneiderMergener, J.; Wenschuh, H. J. Org. Chem. 2001, 66, 507-513. (b) Jobron, L.; Hummel, G. Angew. Chem., Int. Ed. 2000, 39, 1621-1624. (c) Ast, T.; Heine, N.; Germeroth, L.; Schneider-Mergener, J.; Wenschuh, H. Tetrahedron Lett. 1999, 40, 4317-4318. (10) For a recent MW review, see: Lidstrom, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron 2001, 57, 9225-9283. (11) Scharn, D.; Wenschuh, H.; Reineke, U.; Schneider-Mergener, J.; Germeroth, L. J. Comb. Chem. 2000, 2, 361-369. (12) Babu, G.; Perumal, P. T. Synth. Commun. 1997, 27, 3677-3682. 2020
SPOT-synthesis have relied on reaction of cellulose with epibromohydrin.7,11 We chose to avoid this toxic, expensive reagent and found direct tosylation of support 18 to be an effective method to activate cellulose prior to introduction of diamine 2 as a “spacer” element. By controlling the concentration of TsCl and reaction time, the loading level of the support could be varied from 50 nmol/cm2 to 10 µmol/ cm2.17 Diamine 2 was installed as a “spacer” since this unit has been shown to improve the accessibility of support-bound molecules for subsequent reactions and on-support assays.11 MW irradiation of tosyl cellulose in neat diamine 2 (300 W) significantly expedited spacer incorporation; support 3 was generated in 15 min as opposed to the 6 h required for similar incorporation using traditional methods (100 °C, in a drying oven). We found MW heating of membranes in shallow glass vessels to be straightforward and reproducible using a commercial multimodal MW reactor.18 In contrast, a household MW oven (Kenmore model 721; 300 W) gave irreproducible results for heating planar support reactions and underscores the importance of using dedicated, commercial MW reactors for synthesis.5,10 We next introduced a versatile acid-cleavable, Wang-type linker system onto amino-cellulose support 3 (Scheme 1).19 (13) Katritzky, A. R.; Serdyuk, L.; Chassaing, C.; Toader, D.; Wang, X.; Forood, B.; Flatt, B.; Sun, C.; Vo, K. J. Comb. Chem. 2000, 2, 182185 and references therein. (14) Powers, D. G.; Casebier, D. S.; Fokas, D.; Ryan, W. J.; Troth, J. R.; Coffen, D. L. Tetrahedron 1998, 54, 4085-4096. (15) Howitz, K. T.; Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.; Lavu, S.; Wood, J. G.; Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang, L. L.; Scherer, B.; Sinclair, D. A. Nature 2003, 425, 191-196. (16) Lawrence, N. J.; Rennison, D.; McGown, A. T.; Ducki, S.; Gul, L. A.; Hadfield, J. A.; Khan, N. J. Comb. Chem. 2001, 3, 421-426. (17) Functionalization levels were determined by quantitation of Fmocderivatized amino-support 3 using UV spectroscopy (at 296 nm). (18) All MW-assisted reactions on cellulose supports were performed in a Milestone Ethos Microsynth multimodal reactor using power (wattage) control. Temperature control was not possible due to the low solvent volumes used. Supports were washed with various solvents and dried routinely between each synthesis step; see the Supporting Information. Org. Lett., Vol. 6, No. 12, 2004
First, 4-formylphenoxyacetic acid (4) was coupled to support 3 via a standard carbodiimide coupling. Aldehyde support 5 was then subjected to NaBH4 to yield benzyl alcohol-derived support 6. Notably, this four-step reaction sequence did not impact the integrity of the cellulose membrane; we routinely generate support 6 on large scale (ca. 300 cm2 sheets) and find this support/linker system to be highly robust for SPOTsynthesis. We evaluated the feasibility of MW-assisted synthesis on support 6 through the generation of select chalcones. First, we attached various hydroxyacetophenones 7 to support 6 via MW-assisted nucleophilic substitution (Scheme 2).
Scheme 2.
Initial Building Block Loading
Support 6 was activated immediately prior to use by treatment with TsCl in DMF. Solutions of acetophenones 7 and base (KO-t-Bu, in DMSO) were then spotted onto activated support 6 in a spatially addressable manner (SPOT size ) 0.28 cm2) and subjected to MW irradiation at 500 W for 10 min.18 Reproducible acetophenone (7) loadings ranging from 100 to 200 nmol/cm2 were obtained via this protocol (as determined after physically punching-out the SPOT and TFA-mediated cleavage).20 This short reaction time contrasts with the 5-10 h required for analogous solid-phase nucleophilic displacements using traditional heating methods (50 °C).13 Detailed optimization work revealed that high-boiling polar solvents, such as DMSO, and concentrated building block solutions (ca. 2.0 M 7) were required for high loadings (data not shown). These conditions allow for sustained MW heating5,10 and were used for all subsequent MW-assisted SPOT-syntheses. We next investigated the feasibility of MW-assisted Claisen-Schmidt condensations in a SPOT format (Table 1). In preliminary experiments, spotting of support 8 with various benzaldehydes and base in polar solvents (e.g., KOH in H2O) followed by MW irradiation at 400 W for 20 min generated chalcone species 10 (after TFA cleavage) with good to excellent conversions (60-98%).18,21 This represents a marked reduction in reaction time and a concomitant enhancement in yield; these condensation reactions typically require ca. 2-48 h for lower conversions using conventional solid-support synthesis methods.13,22 With these two reactions optimized, we synthesized a test library of chalcones on support 6 (Table 1). Six acetophe(19) Wang, S. S. J. Am. Chem. Soc. 1973, 95, 1328-1333. (20) Compounds were cleaved quantitatively from the Wang linker by treatment with TFA vapor (in a vacuum desiccator, 25 °C, 60 min). See refs 11 and 19. (21) Both cis and trans chalcone isomers were observed in certain cases; see the Supporting Information. (22) Control SPOT reactions (30 min, 25 °C) generated chalcones in low yields (ca. 10%); see the Supporting Information. Org. Lett., Vol. 6, No. 12, 2004
Table 1. Chalcone Test Library Synthesized on Support 6
entrya
R1 (7)
R2 (9)
purityb (%)
10a 10b 10c 10d 10e 10f 10g 10h 10i 10j 10k 10l 10m 10n 10o 10p 10q 10r 10s 10t 10u 10v 10w 10x 10y 10z 10a′ 10b′ 10c′ 10d′ 10e′ 10f ′ 10g′ 10h′ 10i′ 10j′ 10k′ 10l′
4-OH 4-OH 4-OH 4-OH 4-OH 4-OH 4-OH 4-OH 3-OH 3-OH 3-OH 3-OH 3-OH 3-OH 3-OH 3-OH 4-OH, 2-Me 4-OH, 2-Me 4-OH, 2-Me 4-OH, 2-Me 4-OH, 2-Me 4-OH, 2-Me 4-OH, 2-Me 4-OH, 2-Me 4-OH, 3-OMe 4-OH, 3-OMe 4-OH, 3-OMe 4-OH, 3-OMe 4-OH, 3-OMe 4-OH, 3-OMe 4-OH, 3-OMe 4-OH, 3-OMe 4-OH, 3-NO2 4-OH, 3-NO2 4-OH, 3-NO2 4-OH, 3-NO2 4-OH, 2-OH 4-OH, 2-OH
3-OH 3-Br 4-Cl 3-OH, 4-NO2 4-OH 4-OH, 3-Br 4-OH, 3-OMe 5-I, 4-OH, 3-OMe 3-OH 3-Br 4-Cl 3-OH, 4-NO2 4-OH 4-OH, 3-Br 4-OH, 3-OMe 5-I, 4-OH, 3-OMe 3-OH 3-Br 4-Cl 3-OH, 4-NO2 4-OH 4-OH, 3-Br 4-OH, 3-OMe 5-I, 4-OH, 3-OMe 3-OH 3-Br 4-Cl 3-OH, 4-NO2 4-OH 4-OH, 3-Br 4-OH, 3-OMe 5-I, 4-OH, 3-OMe 3-OH 3-Br 4-Cl 3-OH, 4-NO2 3-OH 3-Br
92 89 99 93 70 69 79 75 90 >99 92 86 84 81 91 90 63 67 90 74 43 62 68 66 95 90 92 96 87 81 84 87 66 65 51 59 40 52
a MW reaction conditions, solvent volumes, and base concentration vary for certain substrates. See text and Supporting Information. b Determined by integration of the HPLC trace at 254 nm.
nones 7 and eight benzaldehydes 9 were selected as library building blocks, based on their solubility in polar solvents and the presence of functional group handles, i.e., hydroxyl and/or halogen, for the incorporation of additional library diversity elements.2b The building blocks were combined to yield a parallel array of 40 unique chalcones (10a-10l′) with good to excellent conversions in almost all cases (as determined after TFA cleavage).20,21 Different basic reaction conditions were required depending on the benzaldehyde species (9). More reactive benzaldehydes, such as 3-hydroxybenzaldehyde, underwent efficient condensation at 1.0 M in 2021
a 6 N aq KOH solution, while less reactive substrates, such as vanillin, required a more concentrated solution of both aldehyde (2.0 M) and base (12 N aq KOH) to achieve good conversion. Preparation of the library in a spatially addressed format permitted the application of different reaction conditions in parallel and expedited library synthesis. Indeed, this library was synthesized in only 60 min (for three reaction steps starting from support 6). These results underscore the utility of MW-assisted reactions in library synthesis.5 To further investigate the scope of MW-assisted synthesis on support 6, we examined several heterocycle-generating reactions on support-bound chalcones.13 We discovered that the condensation of chalcones 11 with either acetamidine or benzamidine hydrochloride (12) yielded dihydropyrimidines 13 with good to excellent conversions under MW-assisted conditions (Table 2).23 We then prepared a small library of
Table 2. Dihydropyrimidines Synthesized on Support 6
entry
R1
R2
R3
puritya (%)
13a 13b 13c 13d 13e 13f 13g 13h 13i 13j 13k 13l
4-OH 4-OH 4-OH 4-OH 3-OH 3-OH 3-OH 3-OH 3-OMe, 4-OH 3-OMe, 4-OH 3-OMe, 4-OH 3-OMe, 4-OH
3-Br 3-Br 4-Cl 4-Cl 3-Br 3-Br 4-Cl 4-Cl 3-Br 3-Br 4-Cl 4-Cl
Me Ph Me Ph Me Ph Me Ph Me Ph Me Ph
88 80 83 69 86 86 73 84 79 76 82 83
a
Determined by integration of the HPLC trace at 254 nm.
12 dihydropyrimidines (13a-l) using MW-assisted SPOTsynthesis. Interestingly, we observed the formation of only trace amounts of the related pyrimidine derivatives under these reaction conditions (30 min, MW 400 W, air atmosphere).18 This is in contrast to previous pyrimidine syntheses on solid-phase (ca. 20 h, 100 °C in oil bath)13 where no dihydropyrimidine intermediates were isolated. These data demonstrate how MW-assisted SPOT methods may complement current solid-phase techniques. Representative LC traces for a three-step sequence of single SPOT reactions (ca. 100 nmol/SPOT) are shown in Figure 2 and reveal the efficient conversions achievable using MW-assisted SPOT-synthesis on support 6. One advantage of the SPOT-synthesis technique is that it provides rapid access to compounds in small quantities (23) The absolute tautomeric form of 13 remains unknown. Dihydropyrimidines can exist as 1,4 or 1,6 annular tautomers, see: Weis, A. L. Tetrahedron Lett. 1982, 23, 449-452. 2022
Figure 2. Overlaid LC traces for crude 4-hydroxyacetophenone, chalcone 10b, and dihydropyrimidine 13a cleaved from individual SPOTs of support 6. UV detection at 254 nm. The smaller peak (*) is the cis isomer of 10b.21
(nanomolar to micromolar scale) that are sufficient for characterization and biological evaluation. The ability to translate SPOT-synthesis protocols directly from membrane supports to another form of cellulose that is amenable to larger scale synthesis would expand the utility of this methodology further. We examined MW-assisted synthesis on cellulose powder, fibers, and Perloza beads24,25 as potential scale-up synthesis methods. We found inexpensive Perloza beads (100 µm diameter) to be mechanically robust and readily functionalized with our Wang-type linker/spacer system using chemistry analogous to that outlined in Scheme 1 for support 6 (ca. 10-80 µmol/g loadings). In initial studies, this alternate support system was compatible with MW-assisted chalcone synthesis,18 yielding chalcone 10e, for example, in good purity (74%) at a loading of 4.3 µmol/ g. We note that large-scale synthesis on Perloza (10-100 g) will require different MW reaction conditions; current work involves evaluation of continuous flow MW methods.5 In summary, we have demonstrated that MW-assisted SPOT-synthesis is a viable approach for the generation of parallel combinatorial libraries. We have developed a robust support/linker system for SPOT-synthesis (6), established its compatibility with a range of MW-assisted organic reactions, and extended this methodology to another cellulose support type for larger scale reactions. This combination of techniques presents an accessible and versatile platform for combinatorial discovery and should allow parallel libraries of moderate size to be prepared and screened on the order of days. Ongoing work is directed at the synthesis and biological evaluation of larger, second-generation chalcone libraries. Acknowledgment. This work was supported by generous start-up funds from UWsMadison and CEM Corp. We thank Prof. Laura Kiessling for helpful discussions and CEM Corp. and Milestone, Inc. for technical assistance with MW synthesis instrumentation. Supporting Information Available: Full experimental details for support derivatization and MW-assisted synthesis. This material is available free of charge via the Internet at http://pubs.acs.org. OL049313F (24) Perloza MT-100 is commercially available from Iontosorb Co. (25) De Luca, L.; Giacomelli, G.; Porcheddu, A.; Salaris, M.; Taddei, M. J. Comb. Chem. 2003, 5, 465-471. Org. Lett., Vol. 6, No. 12, 2004
